best immunogen, injctn. schedule for rabbit
Paul A Bucciaglia
bucc0003 at gold.tc.umn.edu
Thu Mar 30 10:27:48 EST 1995
here are my bottom line questions:
1) which form of a protein would make a better immunogen-
insoluble aggregates in an aqueous buffer (no denaturant), or
electroeluted protein from an SDS-PAGE, with excess SDS electro-dialzyed
out of the prep (protein is largely soluble)?
2) What do folks think of this injection schedule (for rabbit):
-primary injection with 0.5 - 1.0 mg protien in 0.5 ml buffer, plus 0.5
ml Freunds complete. 6-8 sub cutaneous injections in the back.
-boost 5-6 weeks later as above, with Freunds incomplete
- bleed 12-14 days later from ear
here's the gory details:
I have overexpressed a 28kD protein in pET28, and purified about 3 mgs
off a Ni column (after lots of empiricle effort!). The protein is
isolated from E. coli inclusion bodies, and requires solubilization in 6M
guanidine-HCl. The column purified protein appears to be pure on
Coomassie stained gels, although there are many small breakdown or
incomplete extension products which make it difficult to determine if
there are smaller molecular weight contaminants. To ensure purity, I
have the option of running preparative SDS-PAGE and electoeluting the
proteins. Its extra work, but I'm willing to do it as previous attempts
at raising anti-rabbit IgG's have generated Ab's to E.coli proteins which
cross react with plant proteins (28 kD protein is from tobacco cDNA).
However, I have read that particulate immunogens elicit strong responses,
and I was concerned that solubilizing in SDS might decrease the response.
I am primarily interested in Abs for Western blotting, but would be
overjoyed if Abs recognized the native protein.
Any comments, suggestions?
Thanks for listening,
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